Crop Genebank Knowledge Base

Regeneration

Regeneration of radish genetic resources

Contributors to this page: CAAS, China (Qiu Yang, Li Xixiang); Bioversity International, Italy (Imke Thormann, Ehsan Dulloo); CGN, Netherlands (Noortje Bas); IPK, Germany (Andreas Börner, Ulrike Lohwasser); AVRDC, Taiwan (Andreas Ebert); USDA, USA (Larry Robertson); NBPGR, India (Chitra Pandey); SASA, UK (George Campbell); University of Warwick, UK (Charlotte Allender).

Before reading the regeneration details for this crop, read the introduction that gives general guidelines to follow by clicking here.

Introduction

Radish (Raphanus sativus L.) is a cross-pollinated, cool-season and annual or biennial vegetable crop. Some cultivars, especially those developed for spring production and of tropical origin have an annual habit. In the first development year biennials develop a fleshy root.and require vernalization to bolt and flower in the second year. Radishes have numerous varieties, varying in size, color and duration of required cultivation time. The period of cold treatment needed to bolt and flower is different among varieties. Temperatures above 32 °C may not only have a negative effect on seed set as the stigma dries out and the pollen may not germinate but also on seed size and percentage of seed germination as the development of the embryo and endosperm will be negatively affected.

Choice of environment and planting season

Climatic conditions

If possible, choose an environment corresponding to the original collection site conditions.
For vegetative growth
- Radish is a cool-weather, short-season vegetable crop. Grows best in cool and moist weather (15-22 °C for leaf, 13-18 °C for root).
For reproductive growth
- Radish flowers initiate (bolt) after vernalization. It is important to expose plants to cool outside temperatures below 15°C for at least 20 days (usually 30~60 days in CAAS).
- Another option is the storage of germinating seeds or radish seedlings with two leaves in a cool room at 2-5°C for 14 to 40 days. In some specific case, the time should be up to 50~60 days.
- Temperature at flowering and seed set is optimal at 25± 3 °C.

Preparation for regeneration

When to regenerate

When seed stocks drop to 1,500 seeds the accession should be scheduled for regeneration. Distribution can continue until seed stock drops to less than 1000 seeds.
When germination is reduced to 80% or to 85% of initial germination (FAO/IPGRI 1994 and ISTA 2008).
Newly introduced, collected or received materials might require regeneration to meet international standards if seed amount and quality are insufficient.

Pre-treatments

Treat seed with fungicides and insecticides or sterilize the seeds using 1% solution of sodium hypochlorite for 10 minutes or hot water at 50°C for 20 minutes, if required.
Wild types are not pre-treated as they are not sown directly into the field.
Use local recommendations as a guide to types and rates of chemical application.

Field selection and preparation

Choose a field in which the previous crop did not belong to the Brassicaceae family.
Fields selected for regeneration should be at an isolation distance of more than 1000 m away from other Brassicaceae crops.
If the materials are planted in an insect proof net house or mesh cage and isolated safely each other, the isolation distance is not required.
Light soils are preferable e.g. sandy loam, silt loam, or soils with high organic matter, with good drainage and deep soils with a pH of 6.0-7.0.
Greenhouse or cold-frames are needed in some places for cultivation to protect the plants from frost.
Mesh cages or sulfite paper bags are needed if more than one variety is regenerated and the physical isolation is not possible.
Some examples:
- SASA: favours the use of polythene tunnels for maintaining isolation, containing pollinators and maximizing plant development in a controlled environment.
- USDA: All regenerations are performed using 12 x 12 x 6 foot mesh covered cages with honeybee five frame nuclear hives introduced at flowering.
- WARGRU: regeneration is carried out in insect-proof cages in an unheated glasshouse. Plants are grown in pots using a standard commercial compost.

Method of regeneration

Methods used

Radish is regenerated using two methods, seed to seed or root to seed.

Seed-to-seed. Sow seeds into a cold-frame for at least 20 days at temperatures below 15°C for natural vernalization. Alternatively, store the germinating radish seeds in a cool room at 2-5°C for 14 to 40 days. Then, the plants are carefully transplanted to the field at least 30 cm apart.
- CGN: seed-to-seed: sow seed in trays in March, transplant the seedlings in small pots and leave them in a cold frame for at least 20 days before planting out in isolation plots.
Root-to-seed. Sow seeds in autumn and pull up the mature fleshy radish roots in winter. Select the healthiest and most typical of the variety in size, shape and colour for seed saving. Store roots in the cold room or bury them underground after defoliation of top parts. In February expose to 9±1°C for at least three weeks. Replant them at a temperature of 25±3°C, at least 30cm apart.

Planting layout, density and distance

For heterogeneous accessions, such as landraces, plant a total of 300 seeds (at least 100 plants are needed) in rows 2-5m long. Adjust the number of rows according to the length of rows and the distance between plants or rows.
For pure lines that are genetically fixed, such as advanced breeding material, plant a total of 100 seeds (at least 30 plants).
Leave at least 45 cm between rows to allow adequate space for inter-row cultivation.
Space plants 30-45 cm apart, sow from 1cm deep recommended for small radishes to 4 cm deep for large radishes, with up to three seeds per hole.
In the greenhouse or cold-frame, sow three seeds per seedling pot or sow in trays. Transplant the radish seedling at 45 × 30 cm apart.
When the area is subject to heavy rainfall and strong winds, the plants need be protected.
Use net cages to protect each accession. Alternatively, leave 1000 m isolation distance between plots where physical barriers are available and 2000 m if in an open landscape.
Plants grown in rows benefit from support when top-heavy with seed.
Example IPK:
- Cultivated radish: two rows of 2 m are directly sown in the field in spring (small radish) or autumn (radish) using 3-4 g of seeds. 25-30 plants are selected in spring or autumn based on morphological characteristics such as root colour, form, taste, consistency and leave form. The selected plants are transferred into pots and placed into a cold house (4-5 °C) during winter to keep them free from frost. In the following spring they are transferred into small greenhouses and seeds are harvested in late summer to early autumn.
- Wild radish types: 50 seeds are sowed onto a tray in Jan-Feb. The small plantlets are thinned into small pots and then transferred into the greenhouse. 2 rows of a total of 20 plants of the bigger types and 30 of the small types are planted in the greenhouse in early April.

Artificial pollination

Radish is cross-pollinated with a self-incompatibility system.
It is pollinated from the same plant during the bud stage to overcome self-incompatibility, when using paper bags
Spray 3-4% NaCl solution to the stigma before the dew dries every morning during the flowering stage to help overcome self-incompatibility.
Use bees, bumble bees or flies with mesh cage.
Some examples from genebanks regarding type of pollinators and timing of introduction to cages:
- AVRDC: Introduces honey bees in each net cage at the onset of flowering
- CGN: Introduces bumble bees or blow flies when about half of the plants is flowering.
- IPK: uses solitary bees for pollination in greenhouses.
- NGB-NBPGR: Introduces honey bees at the onset of flowering
- SASA: uses polytunnels with bumble bees introduced when about 75% of the plants are flowering.
- USDA: introduces common honey bee five frame nuclear hives at flowering.
- WARGRU: blowflies used as pollinators, introduced when first flowers open and further flies are added as required until flowering has ceased.
Manual pollination by a bamboo pole of one end wrapped with absorbent cotton for each accession in different mesh enclosures under a big house of machine-woven net fabric with meshes

Labelling

Label or tag each row. Use weather-resistant labels and ink.
If using polythene tunnels, each tunnel must be clearly numbered. As with outside labels, graphite pencil is preferable to ink as ink bleaching can occur in a short time when exposed to sunlight.

Crop management

Irrigation

Keep soil moist for uninterrupted growth and best quality.
Peak demand during rapid growth and development stage.
SASA: reduces to almost no irrigation at pre-harvest yellow pod stage

Fertilization

Keep the soil mineral and nutrient content steady for normal plant growth and flower production.
Apply a fertilizer balanced for nitrogen, phosphorus and potassium, based on location conditions, practices, soil test results and fertilizer availability, if necessary.
Example CAAS:
- Apply 2000-3000 kg of farmyard manure, 15 kg of diammonium phosphate, 10 kg of urea per 667 m2 and incorporate it before last ploughing.
- A second application of 10-15 kg of urea per 667 m2 two weeks after planting encourages quick growth.
- Apply 20-30 kg of Compound Fertilizer, 20N-20P-20K at early flowering stage.

Common pests and diseases

Contact plant health experts to identify the symptoms of the likely pests and diseases and the appropriate control measures.
The following are common pests and diseases for radish:

Viruses

Turnip mosaic virus (TuMv)
Cucumber mosaic virus (CMV)
Cauliflower mosaic virus (CaMV )
Radish enation mosaic virus (REMV)

Bacteria

Black Rot (Xanthomonas campestris pv. campestris)
Bacterial leaf spot, Peppery leaf spot (Pseudomonas syringae pv. maculicola)

Fungi

Alternaria leaf spot (Alternaria brassicae, A. brassicicola, and A.raphani)
Black leg (Leptosphaeria maculans, anamorph Phoma lingam)
Black root (Aphanomyces raphani)
Cercospora leaf spot (Cercospora cruciferarum and C. atrogrisea)
Fusarium yellows or Fusarium wilt of radish (Fusarium oxysporum f. sp. raphani)
Downy mildew (Peronospora parasitica)
Clubroot (Plasmodiophora brassicae)
Rhizoctonia scurf (Rhizoctonia solani)
Sclerotinia (Sclerotinia sclerotiorum)
White Rust (Albugo candida)

Insects

Cabbage maggot (Delia radicum)
Cabbage seedpod weevil (Ceutorhynchus obstrictus)
Cabbage aphid (Brevicoryne brassicae)
Turnip aphid (Lipaphis erysimi)
Green peach aphid (Myzus persicae)
Diamondback moth (Plutella xylostella)
Cabbage Looper (Trichoplusia ni)

Others

Nematodes (Meloidogyne spp., Heterodera spp., Ditylenchus dipsaci)

Pest and disease control

Preventive

Seed treatment
Field crop rotation
Herbicide spray before sowing
Sterilizing soil
Deep ploughing
Barrier system
Prevent from slugs through pellets
Remove and destroy all previous crop debris.

Non-preventive

Identify weeds, pests, pathogens in time by inspection. Invite pathologists and virologists to the radish field during the growing season.
Loose soil during early stages of crop growth.
Get rid of off-types within the accession; eliminate unhealthy and dead leaves and roots during the growing season.
Spray with appropriate fungicides, herbicide and insecticide.

Harvesting

Before harvest, prepare mesh or paper harvest bags labeled with the genebank code number and plot identification number.
- Example for treatment before harvest: Pre-harvest, SASA will gently lift, detach from the soil and replace each swollen root by hand to prevent further soil-based moisture entering the plant. The stems are supported by strings stretched between posts. In this condition, the plants can be left until all pods ripen and dry evenly.
Harvest when most or all siliques are dry. Examples for harvest procedures
- AVRDC/NBG-NBPGR/USDA: Harvest is done when all siliques are dry and the inflorescence is cut and stored in nylon mesh bag.
- CGN: Harvest is done when a large number of dry siliques are observed. The top with unripe siliques is cut off; the inflorescence is cut from the plant and placed in a linen sac.
- IPK: Cultivated radish is harvested when the whole plant is dry. The whole plant is cut and put into a linen sac. Siliques of wild plants are harvested in paper bags, rarely in linen sac, when all pods are dry, including those that have already been scattered and lie on the ground. If necessary harvest is carried out in several moments.
- SASA: plants are harvested when all pods are dry. Plant stems are cut below seed-pod level and packed in a large polypropylene self-supporting harvest bag. Most seed collects in the bottom of the bag and the remainder is machine-threshed and winnowed directly off the stem.
- WARGRU: pods from each individual plant are harvested into separate paper bags, labeled with the accession and plant number. Aim to collect seed from a minimum of 40 individually labeled plants.
Record the numbers of plants harvested
Check that the information on the bag label and plot tag are the same. Place the seed harvested from a plot and the plot tag into the labeled harvest bag.
The radish pods need to be dried further under sunshine, in a glasshouse or a drying cabinet for a few days after harvesting.
Examples for drying:
- AVRDC: nylon mesh bags are placed under shade for drying before threshing.
- CGN: Linnen sacs are placed in a drying cabinet where the seeds are dried by forced heated air at about 25 °C for a few days. The sacs are then stored in room at 13°C and 30% RH until the seeds are cleaned in winter.
- IPK: The harvested radish is placed in a drying room at 22±2°C and 20±3% RH until they are dry enough for threshing and cleaning.
- NGB-NBPGR: Harvested siliquaes are placed under shade or in a drying room until the material is dry enough for threshing and cleaning.
- USDA: nylon mesh bags are placed in a forced air drying room for drying before threshing.
- WARGRU: paper bags are placed in a drying room (15% RH, 15°C) until the material is dry enough to thresh.

Post-harvest management

After the radish siliques have been dried in the sun or in a drying cabinet, they need to be broken to release the seed by beating or threshing.
Check the labels of the seed inside and outside of the seed bag or the seed envelope to ensure the identity.
Threshing can be done by stomping on, beating or driving over the pods. USDA uses for example a rubber roller belt thresher to break the siliques and release the seeds.
Wild radish siliques are difficult to thresh. When siliques cannot be removed, the seeds are conserved in the siliques and the siliques are broken into single pieces.
After threshing remove all debris, separate empty seeds and other light material with sieves, a seed cleaner or by blowing or hand cleaning.
Manually separate and discard any visually shriveled, discolored, infected, damaged, empty or germinated seeds.
Place the seeds of the same accession into one paper bag labeled on the inside and outside.
- WARGRU: seed from each individual plant is weighed and recombined in proportion in a single paper bag.
If seed quantity is below threshold and/or obtained from too few plants, reschedule the accession for a further regeneration from the original seed.
Dry the seeds to 3–7% moisture content for medium-term and long-term conservation using a dehumidified drying room. Depending on seed size, drying takes a minimum of 2-4 weeks.
The seed samples should be kept in a paper, mesh or cotton bag on mesh racks during the drying period.
All seed samples are dried to equilibrium in a controlled environment of 5-20 °C and 10-25 % RH.
Examples of drying conditions used in genebanks:
- AVRDC: 15% RH and 20 ºC for at least 4 weeks
- CAAS: 13-30% RH and 15-25 ºC
- CGN: 15% RH and 15ºC for at least 6 weeks
- IPK: 10% RH and 18 ºC, 2-3 weeks.
- NGB-NBPGR: 15%RH and 15ºC for up to four weeks. Seeds received with high moisture content are pre-dried in a warm dry place before transferring into walk-in seed drying rooms or seed drying cabinets
- SASA: 15%RH and 15ºC for up to four weeks.
- USDA: 20% RH and 5oC; drying for 2 months
Prepare several sets of the seed samples for preservation in active, base and safety duplicate collections. Send a sample of each accession to a seed health laboratory for quarantine requirements.
Determine 1000 seed weight, and take sample for viability testing of the seeds and record the results according to standard germination methods (ISTA, 2008). If viability is above threshold level, proceed to storage, if viability is lower, reschedule the accession for a further regeneration from the original seeds.
Pack seeds in airtight containers for conservation and distribution.
Store seeds in the genebank at 4°C and 30% RH in active storage or at -18 to -20°C in long-term storage.
In some genebanks all seeds are stored at temperatures below 0°C: USDA stores active and base collections at -20°C in sealed aluminum foil packets; SASA stores all radish seeds at -22°C.

Monitoring accession identity

Comparisons with previous passport or morphological data

Verify accession identity using leaf, flower, pod and seed traits during the planting and post harvest.

Documentation of information during regeneration

The following information should be collected during regeneration:

Regeneration site
Name of collaborator
Plot reference
Accession number
Source of seed
Preparation of planting materials (pre-treatments)
Sowing date
Field layout used
Environmental conditions ((latitude, longitude, altitude, precipitation, soil type, others)
Field management details (watering, fertilizer, weeding, abnormalities recorded, others)
Germination in the field or greenhouse
Agronomic evaluation; agro-morphological traits recorded
Number of plants established
Days from sowing to flowering
Breeding system
Pollination control method used
Harvest date
Number of plants harvested
Quantity of seeds harvested
Viability of seeds harvested (germination rate)

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References and further reading

Ara N, Ali MO, Ali MM, Basher MK. 1999. Effects of spacing and fertilizer levels on yield and quality of radish seed. Bangladesh journal of scientific and industrial research. 4(2): 73-178.

Breese EL. 1989. Regeneration and multiplication of germplasm resources in seed genebanks: The scientific background. Html version available from: http://www2.bioversityinternational.org/publications/Web_version/209/. Date accessed: 9 June 2010.

Engels JMM, Visser L, editors. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No. 6. IPGRI, Rome, Italy. Available here.

FAO/IPGRI. 1994. Genebank standards. Food and Agriculture Organization of the United Nations, Rome and International Plant Genetic Resources Institute, Rome. Available in English, Spanish, French and Arabic .

FAO/IPGRI. 2001. Multi-Crop Passport Descriptors. FAO and IPGRI, Rome, Italy. Available in English, French and Spanish.

George RAT. 2009. Vegetable seed production. 3rd edition. CABI, UK.

IBPGR. 1990. Descriptors for Brassica and Raphanus. International Board for Plant Genetic Resources, Rome, Italy. Published in association with the Commission of the European Communities. ISBN 92-9043-184-9. Available here (1.5 MB).

ISTA. 2008. International Rules for Seed Testing. Edition 2008. International Seed Testing Association, Bassersdorf, Switzerland.

Kalb T, Sukprakarn S, Juntakool S and Huang Rk. 2006. Saving Seeds of Radish:Saving your own vegetable seeds—a guide for farmers. Publication No. 06-668. The World Vegetable Center; Taiwan

Lovato A, Montanari M, Miggiano A. 1994. Nitrogen fertilization of seed radish (Raphanus sativus L.): effects on yield and N-content in seed, plant and soil. International Symposium on Agrotechnics and Storage of Vegetable and Ornamental Seeds. ISHS Acta Horticulturae 362. ISSN: 0567-7572, Bari, Italy.

Navazio J. Principles and practices of organic radish seed production in the Pacific Northwest. Organic Seed Alliance. Supporting the ethical development and stewardship of seed PO Box 772, Port Townsend, WA 98368.

Peterson C. 1999. Radishes: Easy to sprout, hard to grow right. [online]. The New York Times, May 2, 1999. Available from: http://www.nytimes.com/1999/05/02/style/cuttings-radishes-easy-to-sprout-hard-to-grow-right.html. Date accessed: 9 June 2010.

Radish [online]. Available from http://www.seedtamilnadu.com/nradish.htm. Date accessed: 9 June 2010.

Tzay Fa Sheen. 1981. Vernalization and flower development in seed vernalization type of radish. Studies on seed production and vernalization of Cruciferous crops in the tropics. Journal of agricultural research of China 30(4):38-47

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